Microbiological quality and safety of unfinished UHT milk at storage time-temperature abuse

Int.J.Curr.Microbiol.App.Sci (2018) 7(3): 2278-2296

International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 7 Number 03 (2018)
Journal homepage:

Original Research Article

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Microbiological Quality and Safety of Unfinished UHT Milk at
Storage Time-Temperature Abuse
A. Siti Norashikin, M.A.R. Nor-Khaizura* and W.I. Wan Zunairah
1

Department of Food Science, Faculty of Food Science and Technology,
Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia

*Corresponding author

ABSTRACT

Keywords
UHT milk,
Unfinished, Storage
temperature,
Storage time

Article Info
Accepted:
20 February 2018
Available Online:

10 March 2018

The objective of this study is to determine the effect of storage time-temperature abuse on
the microbiological quality and safety of unfinished UHT milk. Therefore, the present
study attempts to imitate the condition of unfinished UHT milk during consumption. The
UHT milk was opened and drank and then the UHT milk was kept at three different
storage temperature of 15 ± 1°C, 25 ± 1°C, 35 ± 1°C for 2, 4, and 6 hours. The
microbiological analysis had been conducted which includes the account of the number of
bacteria regarding Total Plate Count (TPC), Yeast and moulds count, Mesophilic
sporeformers count, Bacillus Cereus, Staphylococcus aureus, Total and Fecal Coliform,
Listeria monocytogenes. At the 35°C storage temperature for 6 hours storage time for
unfinished UHT milk, results showed mean of TPC 7.91 log 10 CFU/mL, Yeast and Moulds
counts 6.84 log10 CFU/mL, Mesophilic sporeformers counts 7.55 log10 CFU/mL, Bacillus
cereus counts 7.73 log10 CFU/mL, Staphylococcus aureus counts 8.30 log10 CFU/mL and
Listeria monocytogenes counts 100 CFU/mL. This indicates that unfinished UHT milk is
not safe to consume at this condition since value of all bacteria counts exceeded the
maximum limit (100 CFU/mL for L. monocytogenes and 5.00 log10 CFU/mL for others)
permitted by Food Act 1983 (Act 281) and Food Regulations 1985 and Netherlands
National Food and Commodities Law. Interestingly, there is no detection of total and fecal
coliform in the sample.

Introduction
The milk demand increase globally due to the
awareness to choose nutritional food in daily
meals. Milk is a nutritious food and suitable
for all range consumer. It is a source of
protein and calcium which important to our
body needs. Milk and dairy products provided
more than 70% of calcium in the US diet
(Ding et al., 2016; Huth et al., 2006). In

Malaysia, „Program Susu 1Malaysia (PS1M)‟
under Ministry of Health Malaysia tend to
increase the awareness and help students in
primary school to get sufficient nutrition by
consuming UHT (Ultra-high temperature)
milk supplied in individual boxes for each
student.
However, milk is a perishable food which
susceptible in rapid spoilage by the action of

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the naturally enzyme and contaminating
microorganisms. Thus, it becomes unsafe to
consume. Foodborne disease will depend on
the extent of food safety control in place
through food production, processing and
distribution keeping food clean, separation of
raw and cooked, and cooking thoroughly,
keeping food at safe temperature and using
safe water and raw materials are some of the
important points especially for safety of food
of humans (Addis and Sisay, 2015). Painter et
al., (2013) stated that foodborne outbreaks
cases associated with the consumption of milk
and dairy products occur each year and an

estimated 6,561,951 annual foodborne
illnesses are attributed to dairy products
caused by a variety of pathogens in the United
States, resulting in an estimated 7464
hospitalisations and 121 deaths. Many food
poisoning cases in Malaysia were reported
was involving foodborne disease after
consuming milk, but the causes are still
unknown. However, one possible reason
could be due to student practices, that prone
to open and drink some of the milk, but not
finish it. The unfinished milk is just left at
room temperature for few hours until they
drink it again.
Ultra High Temperature (UHT) processing
heats the milk at a temperature of 138°C for a
few seconds destroys all microbes present in
milk as well as inactivates all the enzymes,
thus gives the milk a better shelf-life and a
more acceptable sensory perception (Bylund,
1995). UHT milk in aseptic packaging is a
shelf stable product. Safety of UHT milk
depends primarily upon ensuring that the
heat-processing is adequate and that container
integrity is maintained (ICMSF, 1978). The
prolong shelf life will secure the industries
and consumer risk toward spoiled products
and foodborne disease. Heat treatment as one
of the processing steps in the manufacturing
of milk that will give an impact to its

microbiological quality before packaged as a
final product.

Milk also contains microflora as the milk
characteristics itself is a suitable medium for
microbial growth. This microflora can induce
the spoilage of milk together with suitable
temperature and time condition. In addition,
presumptive bacteria that are alive and able to
grow in milk are Staphylococcus aureus,
Escherichia coli, Listeria monocytogenes,
Clostridium and Bacillus cereus. Several
outbreaks of Listeriosis have been associated
with contaminated food such as, vegetables,
dairy products as soft cheeses, pasteurised
milk and meat products, on which L.
monocytogenes can multiply even at low
temperatures (Chaturongakul and Boor, 2006;
Consuelo et al., 2009). Besides, C. pefrigens
and B. cereus both can survive the heat
treatment.
Storage temperature and time together with
pH will greatly influence the survival and
growth of microorganisms. Microbial growth
in the milk that is shelf stable for many
months also can be influenced by factors such
as moisture content, pH, processing
parameters, and temperature of storage
(Ledenbach and Marshall, 2010). There are
researches on milk spoilage, and the factors

contribute to the spoilage for raw milk
(AbdElrahman et al., 2013; Schmidt et al.,
2012). Nonetheless, there is a research gap for
the
effect
of
microbiological
and
physicochemical quality of unfinished UHT
milk after being susceptible to the favourable
condition. Therefore, this study was done in
order to determine the effect of storage timetemperature abuse on microbiological quality
and safety of unfinished UHT milk.
Materials and Methods
Samples
The commercial UHT milk was purchased.
Each sample contains 200 mL of UHT milk.
Imitation of unfinished milk followed by
storage at certain temperature and time on the

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milk sample was conducted at Food
Microbiology Laboratory, Faculty of Food
Science and Technology, Universiti Putra
Malaysia.
The unfinished sample was defined by the

unfinished milk, which was opened and drank
by the one person.
Experimental Design
The unfinished milk was stored at three
temperatures (15, 25 and 35°C) for three
storage time (2, 4 and 6 h). The samples were
conducted and analyzed within 12 hours. The
interactions between the microbial growth of
bacteria and pH with three different storage
temperatures and three different storage times
were analysed. All analysis was conducted by
independent triplicated. Each replicated
represents nine boxes of milk samples.
Microbiological analysis
UHT milk samples were analyzed using
standard procedures (APHA, 2001). A 25 mL
of unfinished UHT milk was aseptically
transferred to a sterile stomacher bag and mix
thoroughly, with 225 mL of sterile 0.1%
peptone water. Appropriate decimal serial
dilutions of the sample were prepared using
the same diluents to 10-7 and spread on
different growth media. Total plate counts
(TPC) were determined using the Plate Count
Agar (PCA) (OXOID), incubated at 37oC for
48 hours. Yeast and mould counts were
determined using the Potato Dextrose Agar
(PDA) (OXOID), incubated at 32oC for five
to seven days. Mesophilic sporeformer counts
were determined using the Dextrose Tryptone

Agar (DTA) (OXOID), incubated at 37oC for
48 hours, after heating the inoculated agar at
80oC for ten minutes to destroy vegetative
cells. Bacillus cereus inoculated using
Bacillus Cereus Selective Agar Base
(OXOID) with Egg Yolk Emulsion, incubated
at 37oC for 48 hours. Staphylococcus aureus

was enumerated using the Baird-Parker Agar
(BPA)(OXOID) with Egg Yolk Tellurite
Emulsion which was incubated at 37oC (IDF
145A:1997) for 48 hours; while total coliform
and fecal coliform conducted by using
MacConkey Agar (OXOID), incubated at
37oC for 48 hours. Listeria monocytogenes
was enumerated using PALCAM Agar Base
(OXOID), incubated at 30oC for 48 hours
(IDF143A:1995) by using Buffered Listeria
Enrichment Broth (OXOID), incubated at
30oC for 48 hours. All results were expressed
as log10 colony forming unit/gram (log10
CFU/mL).
Determination of pH
Methods used for the determination of pH
were adopted from the Microbiological
Laboratory Guidebook of USDA/FSIS (Dey
and Lattuada, 1998).
The pH meter (Mettler Toledo Seven Multi
pH) was warmed up before measuring the
sample. The calibration of this pH meter is

conducted by using buffered solutions pH
4.00 and pH 7.00. Then a sample is prepared
in sterile 25mL stork bottle. The electrode of
the pH meter was rinsed and blotted. After
that, the electrode was immersed in the
sample. The pH reading for the sample
measured was recorded after the pH meter
was stabilized for one minute. The means of
the two measurements were recorded.
Measurement of pH for the sample is repeated
in triplicate.
Statistical analysis
All data collected were analyzed using the
Minitab 16 statistical software (MANITAB
Inc., State College, PA), using two-way
analysis of variance (ANOVA) to identify the
significant differences between factors in the
present study. Thus, all the data reported were
the means of triplicates.

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this critical condition as gelation of milk
started (by observation).

Results and Discussion
Microbiological quality and safety of

unfinished UHT milk at different storage
temperature and time
Total plate count, yeast and moulds count and
mesophilic sporeformers count of the UHT
milk (control) were 4.48 ± 0.25; 4.43 ± 0.21
and 4.32 ± 0.10 log10 CFU/mL (Fig. 1),
respectively. Bacillus cereus, Staphylococcus
aureus, Total and Fecal Coliform and Listeria
monocytogenes were not detected. The
microbiological quality of the unfinished
UHT milk with different storage temperature
and time was tabulated in Table 1.
The microbial load of yeast and moulds in
UHT milk was in contrast with the finding
from the study by Al-Tahiri (2005), who
reported absent of yeast and moulds in their
UHT milk samples (Gamal et al., 2015).
Microbial load of the tested sample may differ
where the UHT milk may come from different
bulk tank and pipelines. Furthermore,
borderline for microbial growth in TPC of
UHT product must be absent (Centre for Food
Safety, 2014). UHT milk should not contain
any viable microorganisms (Carl and Mary,
2014). Contamination during the UHT milk
processing could be the reason for the present
of microorganisms in the end product.
Table 1 shows the microbiological quality and
safety of unfinished UHT milk at three
different temperatures and three storage time.

The findings reveal an increase of bacteria
counts at different storage temperature and
time. As expected, there are a higher number
of microbial loads at the 35°C storage
temperature for 6 hours storage time of
unfinished UHT milk tested. This explains
that the unfinished UHT milk is not safe to
consume when it stored (or left) at 35°C for 6
hours. The unfinished UHT milk turns to be
slimy, viscous and fermented off-flavour at

Total Plate Count (TPC) of unfinished UHT
milk at 15°C for 2, 4, and 6 hours were 4.56 ±
0.42; 4.85 ± 0.59 and 6.24 ± 0.34 log10
CFU/mL, at 25°C for 2, 4, and 6 hours were
6.05 ± 1.04; 5.97 ± 0.50 and 7.54 ± 0.86 log10
CFU/mL, at 35°C for 2, 4, and 6 hours were
5.27 ± 0.59; 6.00 ± 0.86 and 7.91 ± 1.11 log10
CFU/mL (Fig. 2), respectively. From the
graph of Figure 2, it shows the microbial
growth increase as storage temperature and
time increase in unfinished UHT milk.
The TPC at 5 and 10°C as stated by Abd
Elrahman et al., (2013) are 2.45 and 2.53
log10 CFU/mL, lower than the values from the
present study. In this study, an increase of
microbial growth of TPC was observed
started at 15°C for 2 hours. Based on the Food
Act 1983 (Act 281) and Food Regulations
1985 (2016), the maximum growth value of

microbiological standard for TPC is 5.0 log10
CFU/mL of heat-treated milk. In this study,
the values of the TPC for the unfinished UHT
milk had exceeded the maximum values
starting from 25°C for 2 hours (6.05 log10
CFU/mL). Koushki et al., (2016) stated that
total microbial count of pasteurised milk on
an expired date is 4.88 log10 CFU/mL.
Interestingly, TPC value of UHT milk at 15°C
in 4 hours (4.85 log10 CFU/mL) shows in
Figure 2 is close to the value of microbial
growth for expired date milk. The growth
value of microbiological standard for TPC
considered acceptable below 5.0 log10
CFU/mL since the sample was opened and
drank.
Although the bacterial count was provided in
this study, the TPC is only used as an
indicator of bacterial populations in
unfinished UHT milk. El-kholy et al., (2016),
stated that most foods especially dairy

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products should be regarded an unsatisfactory
when a large number of microorganisms
present even though these organisms are not

known to be pathogenic. They also stated that
high
aerobic
plate
counts
indicate
contaminated raw materials, unsatisfactory
processing from a sanitary point of view or
cross-contamination in milk. Specific
microbiological testing was needed on
pathogenic and spoilage bacteria of the
sample. On the other hand, microbial growth
regularly increased as storage temperature and
time increased for TPC.
Yeast and moulds count of unfinished UHT
milk at 15°C for 2, 4, and 6 hours were 4.99 ±
0.69; 5.06 ± 0.39 and 7.15 ± 0.77 log10
CFU/mL, at 25oC for 2, 4, and 6 hours were
4.60 ± 0.53; 5.54 ± 0.52 and 7.31 ± 0.39 log10
CFU/mL, at 35oC for 2, 4, and 6 hours were
4.50 ± 0.28; 5.56 ± 1.02 and 6.84 ± 0.44 log10
CFU/mL (Fig. 3), respectively. Yeast and
mouldscount start to increase at 15°C for 4
hours (5.06 log10 CFU/mL) as presented in
Figure 3. It explained that yeast and moulds
able to survive at 15°C and required 4 hours
after opened and drank to grow under the
same temperature. Thus, it is not safe for
consumption as related to the Food Act 1983
(Act 281) and Food Regulations 1985 (2016).

Presumptive yeast and moulds identified
(based on morphology) in the present study
are Saccharomyces cerevisiae, Hericium
corolloides, Penicillium spp., Aspergillus
niger, Geotrichum candidum, Fusarium spp.,
Rhizopus stolonifer and Rhizopus spp., and
Aspergillus flavus as referred in the study by
Pitt and Hocking, (2009).
Fusarium oxysporum is found in flavoured
UHT milk in Australia owing to the
production of thickly walled Chlamydo
conidia and the ability to tolerate low oxygen
tensions (Sørhaug, 2011). Aspergillus spp.
and Penicillium spp. can grow in milk results
from poor sanitation in the processing plant

and entry of mould spores from crosscontamination (Hubert, 2014). Yeasty and
fermented off-flavours and gassy appearance
are often detected when yeast grow to 5.0 to
6.0 log10 CFU/mL (Ledenbach and Marshall,
2010). In Figure 3, yeast and moulds count
slightly increased as storage temperature and
time increased in unfinished UHT milk.
Mesophilic sporeformers count of unfinished
UHT milk at 15°C for 2, 4, and 6 hours were
4.54 ± 0.56; 5.20 ± 0.28 and 7.04 ± 0.50 log10
CFU/mL, at 25oC for 2, 4, and 6 hours were
4.49 ± 0.014; 5.56 ± 0.69 and 7.19 ± 0.59
log10 CFU/mL, at 35°C for 2, 4, and 6 hours
were 4.37 ± 0.52; 5.73 ± 0.98 and 7.55 ± 0.22

log10 CFU/mL (Fig. 4), respectively.
The value of mesophilic sporeformers count
(5.20 ± 1.36 log10 CFU/mL) at 15°C for 4
hours exceeding the maximum limit stated by
European Union (EU) standards. EU
standards for the total count of mesophilic
sporeformer in milk are ≤ 5.0 log10 CFU/mL
(Samaržija et al., 2012). In this study, the
microbial growth of mesophilic sporeformers
exceeding the limit starting at 15°C for 2
hours. This explains the existed mesophilic
sporeformers in UHT milk survived during
UHT processing and increased in microbial
growth when exposed to a favourable
condition. Moreover, cross-contamination had
occurred and increased microbial load in
samples.
Spore-forming bacteria that are present in
milk are important because the formation of
the spore by the bacterium allows it to be
resistant to heat, freezing, chemicals, and
other adverse environments that milk had
undergoes during processing and preparation
(Cousin, 1989). In Figure 4, mesophilic
sporeformers count increased as storage
temperature and time increased in unfinished
UHT milk. As stated in a study by Set low
(2003), spores will remain dormant until the
conditions become favourable for the change.

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Table.1 The microbiological quality and safety (Total Plate Count, Yeast and Moulds, Mesophilicspore formers,
Bacillus cereus, Staphylococcus aureus, Total and Fecal Coliform and Listeria monocytogenes) (log10 CFU/mL)
of unfinished UHT Milk at different storage time-temperature abuse
Microbial Profile

Total plate count

Yeast and moulds count

Mesophilicsporeformers
count

Bacillus cereus

Staphylococcus aureus

Total and fecal coliform

Listeria monocytogenes

Time (Hour)

2
4
6

2
4
6
2
4
6
2

15
4.56±0.42Ba
4.85±0.59Ba
6.24±0.34Aa
4.99±0.69Ba
5.06±0.39Ba
7.15± 0.77Aa
4.54±0.56Ba
5.20±0.28Ba
7.04±0.50Aa
4.48±0.000Ba

4
6
2
4
6
2
4
6

4.84±0.77ABa

6.62±0.83Aa
0.00±0.00Aa
0.00±0.00Ab
8.15±0.21Ba
N/D
N/D
N/D

2
4
6

N/D
N/D
N/D

A-B
a-b

Log10 CFU/mL
Temperature (oC)
25
6.05±1.04Ba
5.97±0.50Ba
7.54±0.86Aa
4.60±0.53Ba
5.54±0.52Ba
7.31±0.39Aa
4.49±0.014Ba
5.56±0.69ABa

7.19±0.059Aa
4.88±0.81Ba

Means with different uppercase superscripts are significantly different (p<0.05) against row
Means with different lowercase superscripts are significantly different (p<0.05) against column

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5.48±1.05Ba
7.68±0.00Aa
4.93±0.11Ba
4.87±0.23Ba
7.53±0.92Aa
N/D
N/D
N/D
CFU/mL
N/D
N/D
N/D

35
5.27±0.59Ba
6.00±0.86ABa
7.91±1.11Aa
4.50±0.28Ba
5.56±1.02Ba
6.84±0.44Aa
4.37±0.52Ba
5.73±0.98ABa

7.55±0.22Aa
4.92±1.29Aa
6.12±0.39Aa
7.73±1.02Ba
4.30±0.00Ba
5.56±0.62Bab
8.30±0.00Aa
N/D
N/D
N/D
100
100
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Table.2 The pH of unfinished UHT Milk at different storage time-temperature abuse.
pH value
Temperature (oC)
25

15
Time (Hour)

Aa

ABa

2

6.50±0.31

6.43±0.0058

4

6.55±0.044Aa

6.44±0.0058Ab

35
6.46±0.10Aa
6.42±0.0057ABb

6
6.52±0.021Aa
6.41±0.010Bb
6.29±0.00Bc
A-B Means with different uppercase superscripts are significantly different (p<0.05) against
row
a-b-c Means with different lowercase superscripts are significantly different (p<0.05) against
column
FIGURES

Fig.1 The microbiological quality and safety of UHT milk (Total Plate Count, Yeast and
Moulds, Mesophilic sporeforemers, Bacillus cereus, Staphylococcus aureus, Total and Fecal
Coliform and Listeria monocytogenes) (log10 CFU/mL).
*Means (SD from seven determinations)

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Fig.2 Total plate count (log10 CFU/mL) of unfinished UHT milk at storage time-temperature of
15, 25 and 35°C for 2, 4 and 6 hours.
*Means (SD from three determinations)

Fig.3 Yeast and moulds count (log10 CFU/mL) of unfinished UHT milk at storage timetemperature of 15, 25 and 35°C for 2, 4 and 6 hours.
*Means (SD from three determinations)

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Fig.4 Mesophilic spore formers count (log10 CFU/mL) of unfinished UHT milk at storage timetemperature of 15, 25 and 35°C for 2, 4 and 6 hours.
*Means (SD from three determinations)

Fig.5 Bacillus cereus (log10 CFU/mL) of unfinished UHT milk at storage time-temperature of
15, 25 and 35°C for 2, 4 and 6 hours.
*Means (SD from three determinations)

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Fig.6 Staphylococcus aureus (log10 CFU/mL) of unfinished UHT milk at storage timetemperature of 15, 25 and 35°C for 2, 4 and 6 hours.

*Means (SD from three determinations)

Fig.7 Listeria monocytogenes (CFU/mL) of leftover UHT milk of unfinished UHT milk at
storage time-temperature of 15, 25 and 35°C for 2, 4 and 6 hours.
*Means (SD from three determinations)

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Fig.8 The pH of unfinished UHT milk at storage time-temperature of 15, 25 and 35°C for 2, 4
and 6 hours.*Means (SD from three determinations)
Meanwhile, several studies have reported that
heat plays an important role in activation of
spores and that its effect varies within species
or even strains (Kim and Foegeding, 1990;
Ghosh et al., 2009; Anzueto, 2014).
Bacillus cereus of unfinished UHT milk at
15°C for 2, 4, and 6 hours were 4.48 ± 0.00;
4.84 ± 0.77 and 6.62 ± 0.83 log10 CFU/mL, at
25°C for 2, 4, and 6 hours were 4.88 ± 0.81;
5.48 ± 1.04 and 7.68 ± 0.00 log10 CFU/mL, at
35°C for 2, 4, and 6 hours were 4.92 ± 1.29;
6.12 ± 0.39 and 7.73 ± 1.03 log10 CFU/mL
(Fig. 5), respectively.
As reported by teGiffel et al., (1996) and
Kumari and Sarkar (2016), the incidence of
higher levels of contamination by B. cereus in
Netherlands for milk were between 1.0 to 4.0

log10 CFU/mL. Thus, the value of B. cereus
count (4.48 ± 1.04 log10 CFU/mL) at 15°C for
2 hours exceeding the maximum limit stated.
This explains unfinished UHT milk was not
safe to consume starting at 15°C for 2 hours
since the microbial growth increased from

this control point. Schmidt et al., (2012)
stated that the pasteurisation step eliminated
all Gram-negative and lactic acid bacteria,
leaving only high G + C Gram-positive and
spore-formers. Presumptive B. cereus which
is Gram-positive and spore-formers bacteria
detected at 15oC from 2 to 6 hours incubation
period range values from 4.48, 4.84 to 6.62
log10 CFU/mL and increased to a maximum
value at 35°C for 6 hours (7.73 log10
CFU/mL) in this study. B. cereus group and
Bacillus subtilis are the most important
spoilage bacteria in dairy environments as
stated by Lücking et al., (2013). They are able
to produce an extracellular enzyme that able
to degrade the quality of milk by reducing the
shelf life of processed milk and dairy products
(Kumari and Sarkar, 2016). They also stated
in their study that heat-resistant spores of B.
cereus could survive heat treatment, be
present in the milk, and able to germinate and
grow during storage. This can lead to offflavor, clotting, and gelation of milk (Chen et
al., 2003; Furtado, 2005; Kumari and Sarkar,

2016). In addition, the presence of B. cereus

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indicates improper cleaning and sterilization
of the UHT-Aseptic Packaging (UHT-AP)
(Scott, 2008). In the study on food poisoning
potential by B. cereus strains from Norwegian
dairies, the strains were highly cytotoxic
when grew at 25°C but less toxic when
entering human body temperature, 37°C.
Thus, these strains considered pose to minor
risk aboutdiarrhoeal food poisoning (Stenfors
et al., 2007) symptoms. This foodborne
disease is caused by intoxication of diarrhoeal
toxins after consumption of unfinished UHT
milk. In Figure 5, presumptive B. cereus was
able to grow up reaching to 8.0 log10 CFU/mL
at 35°C for 6 hours which is not safe for
human consumption. Magalhães da Veiga
Moreira et al., (2016) reported that yeast, B.
cereus, and Bacillus spp. are commonly
isolated from fermentation of cocoa.
Staphylococcus aureus of unfinished UHT
milk at 15oC for 2, 4, and 6 hours were 0.00 ±
0.00; 0.00 ± 0.00 and 8.15 ± 0.21 log10
CFU/mL, at 25°C for 2, 4, and 6 hours were

4.93 ± 0.12; 4.87 ± 0.23 and 7.53 ± 0.92 log10
CFU/mL, at 35°C for 2, 4, and 6 hours were
4.30 ± 0.00; 5.56 ± 0.62 and 8.30 ± 0.00 log10
CFU/mL (Fig. 6), respectively. S. aureus
shows a significant difference of its microbial
growth in both storage temperature and time.
Several studies have considered 5.0 log10
CFU/mL as the threshold of concern for S.
aureus and its concentration more than 5.0
log10 CFU/mL are unacceptable as state in
FDA (1992), Rho and Schaffner (2007) and
Ding et al., (2016) since S. aureus able to
produce enterotoxin in food in that
population. At 35°C for 4 hours (5.56 log10
CFU/mL), the S. aureus count indicates the
unfinished UHT milk is not safe for
consumption. Fujikawa and Morozumi,
(2006) and Ding et al., (2016), reported that
S. aureus enterotoxin was detectable if the
organism concentration was greater than 6.5
log10 CFU/mL and the temperature higher
than 15°C.

In Figure 6, the present study shows the
concentration of S. aureus higher than 6.50
log10 CFU/mL at 35°C after 6 hours storage
time (8.30 log10 CFU/mL). Based on the study
reported from Ding et al., (2016), the mean of
S. aureus slightly increased during storage
period even after heat treatment had applied

during processing as compared to the S.
aureus concentration before processing.
Microbial count in this product is important
since this is Ready-To-Drink (RTD) dairy
food where there is no heat treatment
followed afterward (Centre of Food Safety,
2014). This can be an issue in microbial
quality and safety to the consumer. The milk
sample was opened and drank (by one person)
for all samples before exposed to the
experimental condition. This contributed to
cross-contamination of S. aureus and other
bacteria from the person and air source.
Mastitis and hygiene of equipment during the
milking (Ninoslava, 2012) can cause the
contamination of S. aureus in UHT milk since
S. aureus is resistant to heat. Thus, the
microbial growth of S. aureus will increase as
temperature and time increased under this
condition. Listeria monocytogenes of
unfinished UHT milk at 15°C for 2, 4, and 6
hours were 0; 0; and 100 CFU/mL, at 25°C for
2, 4, and 6 hours were 0; 0; and 100 CFU/mL,
at 35°C for 2, 4, and 6 hours were 0; 0; and
100 CFU/mL (Fig. 7), respectively. L.
monocytogenes, a pathogen of concern,
causing foodborne disease grows rapidly in
chocolate milk at 13°C as reported by Pearson
and Marth, (1990) and Kotzekidou et al.,
(2008). UHT-processing of milk can stop the

microbial activity of the bacteria but could not
completely kill the pathogenic bacteria. The
increasing of microbial growth can be
observed in the TPC, yeast and moulds,
mesophilic spore formers count, B. cereus,
and S. aureus. But for L. monocytogenes, the
growth is not increasing as temperature and
time increased.

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They grow at 1.0 log10CFU/mL and
stagnantly grow in the samples without any
multiplication of colonies even though the
serial dilution decreased to 10-4, as well as
storage time and temperature increased. The
present study shows presumptive L.
monocytogenes positively detected at 35°C for
2, 4 and 6 hours. As state by Chaturongakul
and Boor (2006), L. monocytogenes can
multiply at low temperatures and able to enter
the carrier animals that shed the organism in
the milk and feces due to the microorganism
resistance to adverse environmental condition
(Chan et al., 2007).
The presence of L. monocytogenes found in
unfinished UHT milk in this study constitutes

a potential hazard for the consumer due to the
storage temperature at 35°C for 2 and 6 hours
holding time. Elliot and Elmer (2007) state
that under the Netherlands National Food and
Commodities Law, L. monocytogenes should
be <100 CFU/mL for all food types except
raw food, based on European Union (EU)
draft regulations.
In RTE foods that can support growth, absent
in five (25 mL) samples are required, unless
the manufacturer can show that numbers will
not exceed 100 CFU/mL throughout the
stated shelf life of the product (IDF, 2013). In
the US, Australia and New Zealand,
regulations
require
absence
of
L.
monocytogenes in five (25 mL) samples in all
cases (IDF, 2013). Limitation of pathogens
bacteria allowable depends on the country
population resistance towards cases fatality
rates.
Determination of pH of unfinished UHT
Milk at different storage temperature and
time
The pH of milk at 15oC for 2, 4, and 6 hours
were 6.50 ± 0.31; 6.55 ± 0.042 and 6.52 ±
0.021, at 25oC for 2, 4, and 6 hours were 6.43

± 0.0058; 6.44 ± 0.0058 and 6.41 ± 0.01, at
35oC for 2, 4, and 6 hours were 6.46 ± 0.10;
6.42 ± 0.12 and 6.29 ± 0.00 (Fig. 8),
respectively. The pH readings of unfinished
UHT milk are optimum for growth since the
values are between pH 6.00 to pH 7.00 (Table
2). The pH of milk normally ranges from 6.4
to 6.8 as reported by Li (2011).
Milk samples should range from pH 6.5-6.7
and sample which out of the pH range
considered acid milk and being rejected (Lai
et al., 2016). The lower the pH, the stronger
the acidity of milk indicated the milk started
to ferment. Storage time and temperature have
a great effect on pH values of the stored
samples as reported by Kocak and Zadow
(1985). Therefore, acidity increases as milk
spoil. Thus, acidity can be quantified to
measure milk quality (Lu et al., 2013).
There was a present of microorganism‟s
growth in Total Plate Count (TPC), yeast and
molds, mesophilic spore formers, Bacillus
cereus, Staphylococcus aureus, and Listeria
monocytogenes in unfinished UHT milk. The
unfinished UHT milk reveals an increasing
trend of microbial growth when stored within
time-temperature abuse. This indicates the
unfinished UHT milk is diminishing in
quality and unsafe to drink.

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How to cite this article:
Siti Norashikin, A., M.A.R. Nor-Khaizura and Wan Zunairah, W.I. 2018. Microbiological
Quality and Safety of Unfinished UHT Milk at Storage Time-Temperature Abuse.
Int.J.Curr.Microbiol.App.Sci. 7(03): 2278-2296. doi: />
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